scholarly journals Applications of Transcriptomics and Proteomics for Understanding Dormancy and Resuscitation in Mycobacterium tuberculosis

2021 ◽  
Vol 12 ◽  
Author(s):  
Manikuntala Kundu ◽  
Joyoti Basu
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Fatty Acid ◽  
Cell Division ◽  
Drug Targets ◽  
Mononuclear Cells ◽  
Clinical Symptoms ◽  
Transcriptional Regulator ◽  
Peripheral Blood Mononuclear ◽  
Latently Infected

Mycobacterium tuberculosis can survive within its host for extended periods of time without any clinical symptoms of disease and reactivate when the immune system is weakened. A detailed understanding of how M. tuberculosis enters into and exits out of dormancy, is necessary in order to develop new strategies for tackling tuberculosis. Omics methodologies are unsupervised and unbiased to any hypothesis, making them useful tools for the discovery of new drug targets. This review summarizes the findings of transcriptomic and proteomic approaches toward understanding dormancy and reactivation of M. tuberculosis. Within the granuloma of latently infected individuals, the bacteria are dormant, with a marked slowdown of growth, division and metabolism. In vitro models have attempted to simulate these features by subjecting the bacterium to hypoxia, nutrient starvation, potassium depletion, growth in the presence of vitamin C, or growth in the presence of long-chain fatty acids. The striking feature of all the models is the upregulation of the DosR regulon, which includes the transcriptional regulator Rv0081, one of the central hubs of dormancy. Also upregulated are chaperone proteins, fatty acid and cholesterol degrading enzymes, the sigma factors SigE and SigB, enzymes of the glyoxylate and the methylcitrate cycle, the Clp proteases and the transcriptional regulator ClgR. Further, there is increased expression of genes involved in mycobactin synthesis, fatty acid degradation, the glyoxylate shunt and gluconeogenesis, in granulomas formed in vitro from peripheral blood mononuclear cells from latently infected individuals compared to naïve individuals. Genes linked to aerobic respiration, replication, transcription, translation and cell division, are downregulated during dormancy in vitro, but upregulated during reactivation. Resuscitation in vitro is associated with upregulation of genes linked to the synthesis of mycolic acids, phthiocerol mycocerosate (PDIM) and sulfolipids; ribosome biosynthesis, replication, transcription and translation, cell division, and genes encoding the five resuscitation promoting factors (Rpfs). The expression of proteases, transposases and insertion sequences, suggests genome reorganization during reactivation.

scholarly journals Effect of Vitamin D3 On the Expression of Antimicrobial Peptide Gene in Experimental Pneumonia in Calf

2021 ◽  
Author(s):  
Parisa Asgharpour ◽  
Zohre Eftekhari ◽  
Mohammad Goli Nadealian ◽  
Gholam Reza Nikbakht Borojeni ◽  
Mohammad Reza Mokhber Dezfouli
Keyword(s):  
Vitamin D ◽  
Treatment Group ◽  
Vitamin D3 ◽  
Pasteurella Multocida ◽  
Mononuclear Cells ◽  
Clinical Symptoms ◽  
Clinical Signs ◽  
Cell Content ◽  
Peripheral Blood Mononuclear

Abstract Background Vitamin D3 has been identified as an immunomodulatory agent that confronts the pathogens via stimulating antimicrobial peptides (AMPs). Objective The effects of vitamin D3 on the expression of AMPs was assessed in experimental pasteurellosis in calves. Methods 10 Holstein crossbred male calves (2–4 months) were chosen and randomly divided into the two groups. Pasteurella multocida was prepared (3×109 CFU/mL) and inoculated in the trachea. Vitamin D3 was injected to the treatment group after confirming the pneumonia. Blood samples were obtained from both groups at different time intervals and the peripheral blood mononuclear cells (PBMCs) were isolated. Clinical symptoms were recorded. Broncho-alveolar lavage was performed to evaluate the lung cell content. On the other hand, 10− 6, 10− 7, and 10 − 8 molar (M) of vitamin D3, was used to evaluate the expression of CD4, BMAP34, and BNBD4 genes using PBMCs under the in vitro conditions. Results The prescription of vitamin D3 to the treatment group caused a decline in clinical signs. Following the vitamin D3 injections the treatment groups under the in vivo conditions, significant increase was observed in the expression level of Defensin (BNBD4), and CD4. Evaluation of bronchoalveolar lavage fluid (BALF) revealed that the amount of neutrophils decreased after vitamin D injection. In vitro, increased expression of Catalicidin (BMAP34), Defensin (BNBD4), and CD4 was observed at a concentration of 10− 6 M of vitamin D3. Conclusion The present study indicated that vitamin D3, exerts immunomodulatory effects on many infectious diseases via activation of VDR pathways and stimulation of AMP production.

CD38 Expression in B-CLL Is Dynamic and Changes Due to Contact with Activating Stimuli Found within the Leukaemic Microenvironment.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2798-2798 ◽  
Author(s):  
Piers E.M. Patten ◽  
Andrea G.S. Buggins ◽  
Julie Richards ◽  
Andrew Wotherspoon ◽  
Terry John Hamblin ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Division ◽  
Mononuclear Cells ◽  
Proliferative Potential ◽  
Activated T Cells ◽  
Peripheral Blood Mononuclear ◽  
Cd38 Expression ◽  
Close Proximity ◽  
Activated T Lymphocytes

Abstract High levels of CD38 expression in B-cell chronic lymphocytic leukaemia (B-CLL) confer a poor prognosis. Although its role in B-CLL is unknown, signalling through CD38 has been implicated in cell survival, trafficking and proliferation. Since proliferation in B-CLL is thought to take place within both bone marrow (BM) and secondary lymphoid tissue, we investigated whether CD38 expression might vary in response to stimuli that occur in these tissue compartments. Firstly, we compared the percentage CD38 expression of CD5/19 cells on 35 paired PB and BM aspirate B-CLL samples. The mean CD38% was significantly higher in BM than PB in all samples (27% vs 19%, p=0.009) including samples with a PB CD38 of 7% or more (33% vs 42%, p=0.047), indicating that factors present in the BM up regulate CD38 expression. Next, CD38 expression and cell division of B-CLL peripheral blood mononuclear cells (PBMCs) were examined in an in vitro system aimed at mimicking the proliferation centre microenvironment where leukaemic cells are situated in close proximity to activated T lymphocytes. Positively selected T cells from 15 B-CLL patients were activated overnight with CD3/28 beads and subsequently cultured with autologous B-CLL PBMCs. Both the percentage of CD19+ CD38+ cells (29.9% vs 59.9%, p=0.003) and CD38 mean fluorescence intensity (75.1 vs 830.8, p=0.005) increased over the 6 day culture period. B-CLL cell division was assessed using the dye carboxyfluorescein diacetate succinimidyl ester (CFSE) in the same co-culture system. This showed that co-culture with autologous activated T-cells can result in B-CLL cell division, and is preceded by CD38 up regulation. In addition, significantly more B-CLL cells underwent at least one division from patients with an initial CD38 level of 7% or more, as compared to under 7% (24.6% vs 10.9%, p=0.031). To further investigate the relationship between B-CLL cell proliferation, CD38 expression and the role of T-cells we examined tissue sections known to contain paraimmunoblasts and other proliferating B-CLL cells. Four colour confocal microscopy using CD3, Ki67, CD38 and CD23 to label frozen B-CLL lymph nodes was employed. Large Ki67+ CD23+ cells were present in close proximity to CD3+ T-cells and these large B-CLL cells had higher CD38 expression than the surrounding small B-CLL lymphocytes. These results support the proposal that CD38 expression in B-CLL is dynamic and may reflect exposure to T-cell derived stimuli which contribute to proliferation in the BM or LN microenvironment. A possible explanation for the poorer prognosis of patients with higher CD38 expression may be that their disease has more proliferative potential.

scholarly journals The ND10 Complex Represses Lytic Human Herpesvirus 6A Replication and Promotes Silencing of the Viral Genome

Viruses ◽  
2018 ◽  
Vol 10 (8) ◽  
pp. 401 ◽  
Author(s):  
Anirban Sanyal ◽  
Nina Wallaschek ◽  
Mandy Glass ◽  
Louis Flamand ◽  
Darren Wight ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Human Herpesvirus ◽  
Virus Genome ◽  
Lytic Replication ◽  
Peripheral Blood Mononuclear ◽  
Latently Infected ◽  
Infected Cells ◽  
T Cell Lines ◽  
Virus Genomes

Human herpesvirus 6A (HHV-6A) replicates in peripheral blood mononuclear cells (PBMCs) and various T-cell lines in vitro. Intriguingly, the virus can also establish latency in these cells, but it remains unknown what influences the decision between lytic replication and the latency of the virus. Incoming virus genomes are confronted with the nuclear domain 10 (ND10) complex as part of an intrinsic antiviral response. Most herpesviruses can efficiently subvert ND10, but its role in HHV-6A infection remains poorly understood. In this study, we investigated if the ND10 complex affects HHV-6A replication and contributes to the silencing of the virus genome during latency. We could demonstrate that ND10 complex was not dissociated upon infection, while the number of ND10 bodies was reduced in lytically infected cells. Virus replication was significantly enhanced upon knock down of the ND10 complex using shRNAs against its major constituents promyelocytic leukemia protein (PML), hDaxx, and Sp100. In addition, we could demonstrate that viral genes are more efficiently silenced in the presence of a functional ND10 complex. Our data thereby provides the first evidence that the cellular ND10 complex plays an important role in suppressing HHV-6A lytic replication and the silencing of the virus genome in latently infected cells.

scholarly journals Increase of CD4+CD25highFoxP3+ cells impairs in vitro human microbicidal activity against Mycobacterium tuberculosis during latent and acute pulmonary tuberculosis

2021 ◽  
Vol 15 (7) ◽  
pp. e0009605
Author(s):  
Lorenzzo Lyrio Stringari ◽  
Luciana Polaco Covre ◽  
Flávia Dias Coelho da Silva ◽  
Vivian Leite de Oliveira ◽  
Maria Carolina Campana ◽  
...  
Keyword(s):  
T Cells ◽  
Mycobacterium Tuberculosis ◽  
Mononuclear Cells ◽  
Critical Role ◽  
Latent Tuberculosis ◽  
Treg Cells ◽  
Host Responses ◽  
Peripheral Blood Mononuclear ◽  
Microbicidal Activity

Background Regulatory T cells (Tregs) play a critical role during Mycobacterium tuberculosis (Mtb) infection, modulating host responses while neutralizing excessive inflammation. However, their impact on regulating host protective immunity is not completely understood. Here, we demonstrate that Treg cells abrogate the in vitro microbicidal activity against Mtb. Methods We evaluated the in vitro microbicidal activity of peripheral blood mononuclear cells (PBMCs) from patients with active tuberculosis (TB), individuals with latent tuberculosis infection (LTBI, TST+/IGRA+) and healthy control (HC, TST-/IGRA-) volunteers. PBMCs, depleted or not of CD4+CD25+ T-cells, were analyzed to determine frequency and influence on microbicidal activity during in vitro Mtb infection with four clinical isolates (S1, S5, R3, and R6) and one reference strain (H37Rv). Results The frequency of CD4+CD25highFoxP3+ cells were significantly higher in Mtb infected whole blood cultures from both TB patients and LTBI individuals when compared to HC. Data from CD4+CD25+ T-cells depletion demonstrate that increase of CD4+CD25highFoxP3+ is associated with an impairment of Th-1 responses and a diminished in vitro microbicidal activity of LTBI and TB groups. Conclusions Tregs restrict host anti-mycobacterial immunity during active disease and latent infection and thereby may contribute to both disease progression and pathogen persistence.

scholarly journals Leishmania spp Epitopes in Humans Naturally Resistant to the Disease: Working Toward a Synthetic Vaccine

2021 ◽  
Vol 11 ◽  
Author(s):  
Magda Melissa Flórez ◽  
Rocío Rodríguez ◽  
José Antonio Cabrera ◽  
Sara M. Robledo ◽  
Gabriela Delgado
Keyword(s):  
Immune Response ◽  
In Silico ◽  
Large Scale ◽  
Mononuclear Cells ◽  
Clinical Symptoms ◽  
Epitope Prediction ◽  
Effective Vaccine ◽  
Peripheral Blood Mononuclear

Vaccines are one of the most effective strategies to fight infectious diseases. Reverse vaccinology strategies provide tools to perform in silico screening and a rational selection of potential candidates on a large scale before reaching in vitro and in vivo evaluations. Leishmania infection in humans produces clinical symptoms in some individuals, while another part of the population is naturally resistant (asymptomatic course) to the disease, and therefore their immune response controls parasite replication. By the identification of epitopes directly in humans, especially in those resistant to the disease, the probabilities of designing an effective vaccine are higher. The aim of this work was the identification of Leishmania epitopes in resistant humans. To achieve that, 11 peptide sequences (from Leishmania antigenic proteins) were selected using epitope prediction tools, and then, peripheral blood mononuclear cells (PBMCs) were isolated from human volunteers who were previously divided into four clinical groups: susceptible, resistant, exposed and not exposed to the parasite. The induction of inflammatory cytokines and lymphoproliferation was assessed using monocyte-derived dendritic cells (moDCs) as antigen-presenting cells (APCs). The response was evaluated after exposing volunteers’ cells to each peptide. As a result, we learned that STI41 and STI46 peptides induced IL-8 and IL-12 in moDCs and lymphoproliferation and low levels of IL-10 in lymphocytes differentially in resistant volunteers, similar behavior to that observed in those individuals to L. panamensis lysate antigens. We conclude that, in silico analysis allowed for the identification of natural Leishmania epitopes in humans, and also STI41 and STI46 peptides could be epitopes that lead to a cellular immune response directed at parasite control.

scholarly journals Tuberculostearic acid (TSA)-containing phosphatidylinositols as reliable marker to determine Mycobacterium tuberculosis bacterial burden

2021 ◽  
Author(s):  
Julius Brandenburg ◽  
Jan Heyckendorf ◽  
Franziska Waldow ◽  
Nicole Zehethofer ◽  
Lara Linnemann ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mononuclear Cells ◽  
Model Systems ◽  
Human Neutrophils ◽  
Bacterial Burden ◽  
Peripheral Blood Mononuclear ◽  
Causative Agents ◽  
Tuberculostearic Acid ◽  
Experimental Model Systems

AbstractIt is estimated that approximately one-fourth of the world's population is infected with strains of the Mycobacterium tuberculosis complex (MTBC), the causative agents of tuberculosis (TB). In this study, we present rationally developed molecular markers for bacterial burden, which are derived from mycobacterial phospholipids. Using lipidomic approaches, we show that tuberculostearic acid (TSA)-containing phosphatidylinositols (PI) are present in all clinically relevant MTBC lineages investigated. For the major abundant lipid PI 16:0_19:0 (TSA), a detection limit equivalent to 102 colony forming units (CFU) was determined for bacterial cultures and approximately 103 for cell culture systems. We further developed a mass spectrometry based targeted lipid assay, which – in contrast to bacterial quantification on solid medium – can be performed within several hours including sample preparation. Translation of this indirect and culture-free detection approach allowed the determination of pathogen loads in infected murine macrophages, human neutrophils and murine lung tissue. We show that marker lipids inferred from the mycobacterial PIs are increased in peripheral blood mononuclear cells (PBMCs) of TB patients beyond the lipid metabolic background in comparison to healthy controls. In a small cohort of drug-susceptible TB patients elevated levels of these marker molecules were detected at therapy start and declined following successful anti-tuberculosis treatment. The concentration of TSA-containing PIs can be used as correlate for reliable and rapid quantification of Mycobacterium tuberculosis (Mtb) burden in experimental in vitro model systems and may also provide a clinically relevant tool for monitoring TB therapy.One Sentence SummaryTuberculostearic acid containing phosphatidylinositols represent a novel, fast to measure, reliable correlate of Mycobacterium tuberculosis bacterial burden in experimental model systems, which makes a future clinical application conceivable.

scholarly journals RNAseq Reveals the Contribution of Interferon Stimulated Genes to the Increased Host Defense and Decreased PPR Viral Replication in Cattle

Viruses ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 463
Author(s):  
Krishnaswamy Tirumurugaan ◽  
Rahul Pawar ◽  
Gopal Dhinakar Raj ◽  
Arthanari Thangavelu ◽  
John Hammond ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Clinical Symptoms ◽  
Small Ruminants ◽  
Control Measures ◽  
Differentially Expressed ◽  
Type I Interferons ◽  
Type I ◽  
Peripheral Blood Mononuclear ◽  
Interferon Stimulated Genes

Peste des petits ruminants virus (PPRV) is known to replicate in a wide variety of ruminants causing very species-specific clinical symptoms. Small ruminants (goats and sheep) are susceptible to disease while domesticated cattle and buffalo are dead-end hosts and do not display clinical symptoms. Understanding the host factors that influence differential pathogenesis and disease susceptibility could help the development of better diagnostics and control measures. To study this, we generated transcriptome data from goat and cattle peripheral blood mononuclear cells (PBMC) experimentally infected with PPRV in-vitro. After identifying differentially expressed genes, we further analyzed these immune related pathway genes using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) and selected candidate genes were validated using in-vitro experiments. Upon PPRV infection, we identified 12 and 22 immune related genes that were differentially expressed in goat and cattle respectively. In both species, this included the interferon stimulated genes (ISGs) IFI44, IFI6, IFIT1, IFIT2, IFIT3, ISG15, Mx1, Mx2, OAS1X, RSAD2, IRF7, DDX58 and DHX58 that were transcribed significantly higher in cattle. PPRV replication in goat PBMCs significantly increased the expression of phosphodiesterase 12 (PDE12), a 2′,5′-oligoadenylate degrading enzyme that contributes to the reduced modulation of interferon-regulated gene targets. Finally, a model is proposed for the differential susceptibility between large and small ruminants based on the expression levels of type-I interferons, ISGs and effector molecules.

scholarly journals Modulation of Gamma Interferon Receptor 1 by Mycobacterium tuberculosis: a Potential Immune Response Evasive Mechanism

2007 ◽  
Vol 75 (5) ◽  
pp. 2500-2510 ◽  
Author(s):  
Amit Singhal ◽  
Anand Jaiswal ◽  
Virendra K. Arora ◽  
Hanumanthappa K. Prasad
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mrna Expression ◽  
Mononuclear Cells ◽  
Surface Expression ◽  
Gamma Interferon ◽  
Mobility Shift ◽  
Down Regulation ◽  
Peripheral Blood Mononuclear ◽  
Ifn Γ

ABSTRACT Mycobacterium tuberculosis inhibits gamma interferon (IFN-γ)-mediated antimycobacterial action by adopting diverse mechanisms. IFN-γ binds to its receptor, IFN-γR, in order to initiate proper signaling. We have observed reduced surface expression levels of IFN-γ receptor 1 (IFN-γR1) in untreated pulmonary tuberculosis patients compared to those in healthy individuals (P < 0.01). Following antitubercular therapy, the expression of IFN-γR1 was restored in these patients. To delineate the mechanism by which M. tuberculosis modulates IFN-γR1, in vitro experiments were designed, wherein the down modulation of IFN-γR1 surface expression was observed for CD14+ cells in peripheral blood mononuclear cells (PBMCs) cocultured with live M. tuberculosis compared to that for uninfected cells (P < 0.01). No modulation of IFN-γR1 expression was observed for CD14+ cells in PBMCs infected with Mycobacterium smegmatis. A time-dependent decrease in IFN-γR1 mRNA expression was observed for PBMCs infected with M. tuberculosis. Similar down modulation of IFN-γR1 protein and mRNA expression in phorbol myristate acetate-differentiated THP-1 cells (pdTHP-1) by M. tuberculosis was observed (P < 0.01). Using reporter gene analysis of 5′ deletion constructs of the IFN-γR1 gene (IFNGR1) promoter, the decrease in IFN-γR1 mRNA in M. tuberculosis-infected pdTHP-1 cells was shown to be due to the decreased transcription of IFNGR1. By immunoblotting and electrophoretic mobility shift assays, the down regulation of stimulating protein 1 (Sp1) expression and its recruitment on the phorbol ester-responsive element of the IFNGR1 promoter in M. tuberculosis-infected pdTHP-1 cells was observed. This down regulation of Sp1 in pdTHP-1 cells cocultured with M. tuberculosis may be responsible for the down regulation of IFN-γR1 expression, thereby potentially altering its receptivity to IFN-γ.

scholarly journals miR-181a Ameliorates the Progression of Myasthenia Gravis by Regulating TRIM9

2021 ◽  
Vol 2021 ◽  
pp. 1-9
Author(s):  
Qiang Wang ◽  
Yunquan Liu ◽  
Shixiang Kuang ◽  
Ruozhao Li ◽  
Ning Weng ◽  
...  
Keyword(s):  
Myasthenia Gravis ◽  
Target Genes ◽  
Mononuclear Cells ◽  
Clinical Symptoms ◽  
Inflammatory Factors ◽  
Peripheral Blood Mononuclear ◽  
Inflammatory Microenvironment ◽  
In Vivo Experiments

Abnormally activated CD4+ T cells are considered to be an important factor in the pathogenesis of myasthenia gravis (MG). In the pathogenesis of MG, the imbalance of proinflammatory cytokines and immune cells maintains the imbalance of immune response and inflammatory microenvironment. Studies have shown that miRNA is involved in the pathogenesis of MG. In our experiment, we extracted peripheral blood mononuclear cells (PBMCs) from MG patients and detected the expression of miR-181a and TRIM9 in PBMCs by qRT-PCR. In vitro experiments were conducted to explore the regulatory mechanism of miR-181a on target genes and its influence on inflammatory factors related to MG disease. Experimental autoimmune myasthenia gravis (EAMG) model mice are established, and the effects of miR-181a on EAMG symptoms and inflammatory factors are explored through in vivo experiments. According to a total of 40 EAMG mice that were successfully modeled, all EAMG mice showed symptoms of muscle weakness; their diet was reduced; their weight gain was slow; and even weight loss occurred. In MG patients and EAMG mice, the expression of miR-181a was low and TRIM9 was highly expressed. Bioinformatics website and dual-luciferase report analysis of miR-181a had a targeting relationship with TRIM9, and miR-181a could target the expression of TRIM9. After upregulating miR-181a or interfering with TRIM9, serum miR-181a in EAMG mice was significantly upregulated; TRIM9 was significantly downregulated; its clinical symptoms were reduced; and the expression of inflammatory factors was reduced. The study finally learned that miR-181a can reduce the level of MG inflammatory factors by targeting the expression of TRIM9 and has the effect of improving the symptoms of MG.

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